The invention relates generally to assays for screening for agents that affect mitochondrial activity. More specifically, the invention is directed to screening methods for use in identifying agents that alter mitochondrial regulation of intracellular calcium. An assay for the presence of extramitochondrial calcium and for factors that influence levels of intramitochondrial and/or extramitochondrial calcium, such as the calcium uniporter (CaUP), is provided herein.
Mitochondria are organelles that are the main energy source in cells of higher organisms. These organelles provide direct and indirect biochemical regulation of a wide array of cellular respiratory, oxidative and metabolic processes, including metabolic energy production, aerobic respiration and intracellular calcium regulation. For example, mitochondria are the site of electron transport chain (ETC) activity, which drives oxidative phosphorylation to produce metabolic energy in the form of adenosine triphosphate (ATP), and which also underlies a central mitochondrial role in intracellular calcium homeostasis. These processes require the maintenance of a mitochondrial membrane electrochemical potential, and defects in such membrane potential can result in a variety of disorders.
In addition to their role in energy production in growing cells, mitochondria (or at least mitochondrial components) participate in programmed cell death (PCD), also known as apoptosis (see Newmeyer et al., Cell 79:353-364, 1994; Liu et al., Cell 86:147-157, 1996). Apoptosis is apparently required for normal development of the nervous system and functioning of the immune system. Some disease states are associated with insufficient apoptosis (e.g., cancer and autoimmune diseases) or excessive levels of apoptosis (e.g., stroke and neurodegeneration). For general reviews of apoptosis, and the role of mitochondria therein, see Green and Reed, Science 281:1309-1312, 1998; Green, Cell 94:695-698, 1998; and Kromer, Nature Medicine 3:614-620, 1997.
Mitochondria contain an outer mitochondrial membrane that serves as an interface between the organelle and the cytosol, a highly folded inner mitochondrial membrane that appears to form attachments to the outer membrane at multiple sites, and an intermembrane space between the two mitochondrial membranes. The subcompartment within the inner mitochondrial membrane is commonly referred to as the mitochondrial matrix (for review, see, e.g., Emster et al., J. Cell Biol. 91:227s, 1981). While the outer membrane is freely permeable to ionic and non-ionic solutes having molecular weights less than about ten kilodaltons, the inner mitochondrial membrane exhibits selective and regulated permeability for many small molecules, including certain cations, and is impermeable to large (greater than about 10 kD) molecules.
Four of the five multisubunit protein complexes (Complexes I, III, IV and V) that mediate ETC activity are localized to the inner mitochondrial membrane. The remaining ETC complex (Complex II) is situated in the matrix. In at least three distinct chemical reactions known to take place within the ETC, protons are moved from the mitochondrial matrix, across the inner membrane, to the intermembrane space. This disequilibrium of charged species creates an electrochemical membrane potential of approximately 220 mV referred to as the xe2x80x9cprotonmotive forcexe2x80x9d (PMF). The PMF, which is often represented by the notation xcex94p, corresponds to the sum of the electric potential (xcex94xcexa8m) and the pH differential (xcex94pH) across the inner membrane according to the equation
xcex94p=xcex94xcexa8mxe2x88x92Zxcex94pH 
wherein Z stands for xe2x88x922.303 RT/F. The value of Z is xe2x88x9259 at 25xc2x0 C. when xcex94p and xcex94xcexa8m are expressed in mV and xcex94pH is expressed in pH units (see, e.g., Emster et al., J. Cell Biol. 91:227s, 1981, and references cited therein).
xcex94xcexa8m provides the energy for phosphorylation of adenosine diphosphate (ADP) to yield ATP by ETC Complex V, a process that is coupled stoichiometrically with transport of a proton into the matrix. xcex94xcexa8m is also the driving force for the influx of cytosolic Ca2+ into the mitochondrion. Under normal metabolic conditions, the inner membrane is largely impermeable to proton movement from the intermembrane space into the matrix, leaving ETC Complex V as the primary means whereby protons can return to the matrix. When, however, the integrity of the inner mitochondrial membrane is compromised, as occurs during mitochondrial permeability transition (MPT) that accompanies certain diseases associated with altered mitochondrial function, protons are able to bypass the conduit of Complex V without generating ATP, thereby uncoupling respiration (i.e., ETC activity) from ATP production. During MPT, xcex94xcexa8m collapses and mitochondrial membranes lose the ability to selectively regulate permeability to solutes both small (e.g., ionic Ca2+, Na+, K+ and H+) and large (e.g., proteins). Loss of mitochondrial potential also appears to be a critical event in the progression of diseases associated with altered mitochondrial function, including degenerative diseases such as Alzheimer""s Disease; diabetes mellitus; Parkinson""s Disease; Huntington""s disease; dystonia; Leber""s hereditary optic neuropathy; schizophrenia; mitochondrial encephalopathy, lactic acidosis, and stroke (MELAS); cancer; psoriasis; hyperproliferative disorders; mitochondrial diabetes and deafness (MIDD) and myoclonic epilepsy ragged red fiber syndrome.
Normal alterations of intramitochondrial Ca2+ are associated with normal metabolic regulation (Dykens, 1998 in Mitochondria and Free Radicals in Neurodegenerative Diseases, Beal, Howell and Bodis-Wollner, Eds., Wiley-Liss, New York, pp. 29-55; Radi et al., 1998 in Mitochondria and Free Radicals in Neurodegenerative Diseases, Beal, Howell and Bodis-Wollner, Eds., Wiley-Liss, New York, pp. 57-89; Gunter and Pfeiffer, 1991, Am. J. Physiol. 27: C755; Gunter et al., Am. J. Physiol. 267:313, 1994). For example, fluctuating levels of mitochondrial free Ca2+ may be responsible for regulating oxidative metabolism in response to increased ATP utilization, via allosteric regulation of enzymes (reviewed by Crompton and Andreeva, Basic Res. Cardiol. 88:513-523, 1993); and the glycerophosphate shuttle (Gunter and Gunter, J. Bioenerg. Biomembr. 26:471, 1994).
Normal mitochondrial function includes regulation of cytosolic free calcium levels by sequestration of excess Ca2+ within the mitochondrial matrix. Depending on cell type, cytosolic Ca2+ concentration is typically 50-100 nM. In normally functioning cells, when Ca2+ levels reach 200-300 nM, mitochondria begin to accumulate Ca2+ as a function of the equilibrium between influx via a Ca2+ uniporter in the inner mitochondrial membrane and Ca2+ efflux via both Na+ dependent and Na+ independent calcium carriers. The low affinity of this rapid uniporter mechanism suggests that the primary uniporter function may be to lower cytosolic Ca2+ in response to pathological elevation of cytosolic free calcium levels, which may result from ATP depletion and/or abnormal calcium influx across the plasma membrane (Gunter and Gunter, J. Bioenerg. Biomembr. 26:471, 1994; Gunter et al., Am. J. Physiol. 267:313, 1994). In certain instances, such perturbation of intracellular calcium homeostasis is a feature of diseases associated with altered mitochondrial function, regardless of whether the calcium regulatory dysfunction is causative of, or a consequence of, altered mitochondrial function including MPT.
In view of the significance of mitochondrial regulation of intracellular calcium and the relationship of this mitochondrial activity to several disease states, there is clearly a need for improved compositions and methods to control mitochondrial calcium homeostasis. To provide improved therapies for such diseases, agents that alter mitochondrial calcium regulation may be beneficial, and assays to specifically detect such agents are needed. The present invention fulfills these needs and further provides other related advantages.
The present invention is directed in part to methods for identifying agents that alter mitochondrial regulation of intracellular calcium. Thus, in one aspect the invention provides a method of identifying an agent that alters mitochondrial function, comprising (a) contacting, in each of a plurality of reaction vessels in a high throughput screening array, (i)a biological sample comprising a cell containing cytosol, a mitochondrion and a calcium indicator molecule, under conditions that permit maintenance of mitochondrial membrane potential, with (ii) a source of calcium cations, wherein the calcium indicator molecule is capable of generating a detectable signal that is proportional to the level of calcium in the cytosol; (b) detecting in each reaction vessel the signal generated by the calcium indicator molecule at a plurality of time points; and (c) comparing the signal generated by the calcium indicator molecule at one or more of the time points in the absence of a candidate agent, to the signal generated by the calcium indicator molecule at one or more of the time points in the presence of the candidate agent, and therefrom identifying an agent that alters mitochondrial function. In one embodiment the step of contacting is repeated at least once. In another embodiment the sample contains at least one compound that alters intracellular distribution of a calcium cation. In a further embodiment the compound that alters intracellular calcium cation distribution is thapsigargin or Ru360. In another embodiment the compound that alters intracellular calcium cation distribution is a calcium ionophore or a membrane permeable compound that alters intracellular calcium distribution. In another embodiment the sample contains at least one compound that uncouples oxidative phosphorylation from ATP production. In another embodiment the candidate agent is membrane permeable, and in another embodiment the calcium indicator molecule is membrane permeable. In another embodiment the source of calcium cations is exogenous to the cell. In another embodiment the sample contains at least one compound that uncouples oxidative phosphorylation from ATP production. In another embodiment the cell comprises at least one polypeptide that is a member of the Bcl-2 family. In another embodiment the cell expresses a gene encoding a polypeptide that regulates cytosolic calcium. In another embodiment the gene encodes a mitochondrial calcium uniporter. In another embodiment the gene is a transfected gene. In another embodiment the gene encodes a mitochondrial calcium uniporter. In another embodiment the cell is a permeabilized cell. In certain embodiments the cell adheres to a solid substrate and in certain other embodiments the cell is a non-adherent cell.
It is another aspect of the present invention to provide a method of identifying an agent that uncouples oxidative phosphorylation from ATP production, comprising (a) contacting, in each of a plurality of reaction vessels in a high throughput screening array, (i) a biological sample comprising a cell containing cytosol, a mitochondrion and a calcium indicator molecule, under conditions that permit maintenance of mitochondrial membrane potential, with (ii) a source of calcium cations, wherein the calcium indicator molecule is capable of generating a detectable signal that is proportional to the level of calcium in the cytosol; (b) detecting in each reaction vessel the signal generated by the calcium indicator molecule at a plurality of time points; (c) repeating steps (a) and (b) at least once; and (d) comparing (i) the signal generated by the calcium indicator molecule at one or more of the time points prior to and following at least one of the contacting steps in the absence of the candidate agent, to (ii) the signal generated by the calcium indicator molecule at one or more of the time points prior to and following at least one of the contacting steps in the presence of the candidate agent, wherein an increased level of calcium in the cytosol at a time point prior to a contacting step in the presence of the agent, compared to the level of calcium in the cytosol prior to a contacting step in the absence of the agent, indicates an agent that uncouples oxidative phosphorylation from ATP production.
In another aspect the invention provides a method of identifying an agent that that is a respiratory inhibitor, comprising: (a) contacting, in each of a plurality of reaction vessels in a high throughput screening array, (i) a biological sample comprising a cell containing cytosol, a mitochondrion and a calcium indicator molecule, under conditions that permit maintenance of mitochondrial membrane potential, with (ii) a source of calcium cations, wherein the calcium indicator molecule is capable of generating a detectable signal that is proportional to the level of calcium in the cytosol; (b) detecting in each reaction vessel the signal generated by the calcium indicator molecule at a plurality of time points; (c) repeating steps (a) and (b) at least once; and (d) comparing (i) the signal generated by the calcium indicator molecule at one or more of the time points prior to and following at least one of the contacting steps in the absence of the candidate agent, to (ii) the signal generated by the calcium indicator molecule at one or more of the time points prior to and following at least one of the contacting steps in the presence of the candidate agent, wherein an increased level of calcium in the cytosol at a time point prior to a contacting step in the presence of the agent, compared to the level of calcium in the cytosol prior to a contacting step in the absence of the agent, indicates an agent that is a respiratory inhibitor.
In another embodiment the invention provides a method of identifying an agent that alters a mitochondrial calcium uniporter, comprising: (a) contacting, in each of a plurality of reaction vessels in a high throughput screening array, (i) a biological sample comprising a cell containing cytosol, a mitochondrion and a calcium indicator molecule, under conditions that permit maintenance of mitochondrial membrane potential, with (ii) a source of calcium cations, wherein the calcium indicator molecule is capable of generating a detectable signal that is proportional to the level of calcium in the cytosol; (b) detecting in each reaction vessel the signal generated by the calcium indicator molecule at a plurality of time points; (c) repeating steps (a) and (b) at least once; and (d) comparing (i) the signal generated by the calcium indicator molecule at one or more of the time points prior to and following at least one of the contacting steps in the absence of the candidate agent, to (ii) the signal generated by the calcium indicator molecule at one or more of the time points prior to and following at least one of the contacting steps in the presence of the candidate agent, wherein an increased level of calcium in the cytosol at a time point following a contacting step in the presence of the agent, compared to the level of calcium in the cytosol following a contacting step in the absence of the agent, indicates that the agent alters a mitochondrial calcium uniporter.
In another embodiment there is provided a method of identifying an agent that alters mitochondrial function, comprising (a) contacting (i) a biological sample comprising a cell containing cytosol, a mitochondrion and a calcium indicator molecule, under conditions that permit maintenance of mitochondrial membrane potential, with (ii) a source of calcium cations, wherein the calcium indicator molecule is capable of generating a detectable signal that is proportional to the level of calcium in the cytosol; (b) detecting the signal generated by the calcium indicator molecule at a plurality of time points; and (c) comparing the signal generated by the calcium indicator molecule at one or more of the time points in the absence of the candidate agent, to the signal generated by the calcium indicator molecule at one or more of the time points in the presence of the candidate agent, and therefrom identifying an agent that alters mitochondrial function. In one embodiment the step of contacting is repeated at least once. In another embodiment the sample contains at least one compound that alters intracellular distribution of a calcium cation. In another embodiment the compound that alters intracellular calcium cation distribution is thapsigargin or Ru360. In another embodiment the compound that alters intracellular calcium cation distribution is selected from the group consisting of a calcium ionophore and a membrane permeable compound that alters intracellular calcium distribution. In another embodiment the sample contains at least one compound that uncouples oxidative phosphorylation from ATP production. In another embodiment the candidate agent is membrane permeable. In another embodiment the calcium indicator molecule is membrane permeable. In another embodiment the source of calcium cations is exogenous to the cell. In another embodiment the sample contains at least one compound that uncouples oxidative phosphorylation from ATP production. In another embodiment the cell comprises at least one polypeptide that is a member of the Bcl-2 family. In another embodiment the cell expresses a gene encoding a polypeptide that regulates cytosolic calcium. In another embodiment the gene encodes a mitochondrial calcium uniporter. In another embodiment the gene is a transfected gene. In another embodiment the gene encodes a mitochondrial calcium uniporter. In another embodiment the cell is a permeabilized cell. In another embodiment the cell adheres to a solid substrate. In another embodiment the cell is a non-adherent cell.
In still another embodiment the invention provides a method of identifying an agent that uncouples oxidative phosphorylation from ATP production, comprising: (a) contacting (i) a biological sample comprising a cell containing cytosol, a mitochondrion and a calcium indicator molecule, under conditions that permit maintenance of mitochondrial membrane potential, with (ii) a source of calcium cations, wherein the calcium indicator molecule is capable of generating a detectable signal that is proportional to the level of calcium in the cytosol; (b) detecting the signal generated by the calcium indicator molecule at a plurality of time points; (c) repeating steps (a) and (b) at least once; and (d) comparing (i) the signal generated by the calcium indicator molecule at one or more of the time points prior to and following at least one of the contacting steps in the absence of the candidate agent, to (ii) the signal generated by the calcium indicator molecule at one or more of the time points prior to and following at least one of the contacting steps in the presence of the candidate agent, wherein an increased level of calcium in the cytosol at a time point prior to a contacting step in the presence of the agent, compared to the level of calcium in the cytosol prior to a contacting step in the absence of the agent, indicates an agent that uncouples oxidative phosphorylation from ATP production.
In another embodiment the invention provides a method of identifying an agent that is a respiratory inhibitor, comprising: (a) contacting (i) a biological sample comprising a cell containing cytosol, a mitochondrion and a calcium indicator molecule, under conditions that permit maintenance of mitochondrial membrane potential, with (ii) a source of calcium cations, wherein the calcium indicator molecule is capable of generating a detectable signal that is proportional to the level of calcium in the cytosol; (b) detecting the signal generated by the calcium indicator molecule at a plurality of time points; (c) repeating steps (a) and (b) at least once; and (d) comparing (i) the signal generated by the calcium indicator molecule at one or more of the time points prior to and following at least one of the contacting steps in the absence of the candidate agent, to (ii) the signal generated by the calcium indicator molecule at one or more of the time points prior to and following at least one of the contacting steps in the presence of the candidate agent, wherein an increased level of calcium in the cytosol at a time point prior to a contacting step in the presence of the agent, compared to the level of calcium in the cytosol prior to a contacting step in the absence of the agent, indicates an agent that is a respiratory inhibitor.
In another embodiment the invention provides a method of identifying an agent that alters a mitochondrial calcium uniporter, comprising (a) contacting, (i) a biological sample comprising a cell containing cytosol, a mitochondrion and a calcium indicator molecule, under conditions that permit maintenance of mitochondrial membrane potential, with (ii) a source of calcium cations, wherein the calcium indicator molecule is capable of generating a detectable signal that is proportional to the level of calcium in the cytosol; (b) detecting the signal generated by the calcium indicator molecule at a plurality of time points; (c) repeating steps (a) and (b) at least once; and (d) comparing (i) the signal generated by the calcium indicator molecule at one or more of the time points prior to and following at least one of the contacting steps in the absence of the candidate agent, to (ii) the signal generated by the calcium indicator molecule at one or more of the time points prior to and following at least one of the contacting steps in the presence of the candidate agent, wherein an increased level of calcium in the cytosol at a time point following a contacting step in the presence of the agent, compared to the level of calcium in the cytosol following a contacting step in the absence of the agent, indicates that the agent alters a mitochondrial calcium uniporter.
In another embodiment the invention provides a method of identifying an agent that alters mitochondrial function, comprising: (a) contacting, in each of a plurality of reaction vessels in a high throughput screening array, (i) a biological sample comprising a cell containing a mitochondrion, cytosol and a calcium indicator molecule, under conditions that permit maintenance of mitochondrial membrane potential, and wherein the calcium indicator molecule is membrane permeable and capable of generating a detectable signal that is proportional to the level of calcium in the cytosol, with (ii) a calcium ionophore, under conditions and for a time sufficient to increase calcium levels within the cell; (b) detecting in each reaction vessel the signal generated by the calcium indicator molecule at a plurality of time points; and (c) comparing the signal generated by the calcium indicator molecule at one or more of the time points in the absence of the candidate agent, to the signal generated by the calcium indicator molecule at one or more of the time points in the presence of the candidate agent, and therefrom identifying an agent that alters mitochondrial function.
In another embodiment the calcium ionophore is ionomycin, A23187, NMDA or a cell depolarization signal. In another embodiment the step of contacting is repeated at least once. In another embodiment the sample contains at least one compound that alters intracellular distribution of a calcium cation. In another embodiment the compound that alters intracellular calcium cation distribution is thapsigargin or Ru360. In another embodiment the compound that alters intracellular calcium cation distribution is a calcium ionophore or a membrane permeable compound that alters intracellular calcium distribution. In another embodiment the sample contains at least one compound that uncouples oxidative phosphorylation from ATP production. In another embodiment the candidate agent is membrane permeable. In another embodiment the source of calcium cations is exogenous to the cell. In another embodiment the sample contains at least one compound that uncouples oxidative phosphorylation from ATP production. In another embodiment the cell comprises at least one polypeptide that is a member of the Bcl-2 family. In another embodiment the cell expresses a gene encoding a polypeptide that regulates cytosolic calcium. In certain further embodiments the gene encodes a mitochondrial calcium uniporter, and in certain other embodiments the gene is a transfected gene. In certain further embodiments the gene encodes a mitochondrial calcium uniporter. In another embodiment the cell adheres to a solid substrate, and in certain other embodiments the cell is a non-adherent cell.
In another embodiment the invention provides a method of identifying an agent that uncouples oxidative phosphorylation from ATP production, comprising (a) contacting (i) a biological sample comprising a cell containing cytosol, a mitochondrion and a calcium indicator molecule, under conditions that permit maintenance of mitochondrial membrane potential, and wherein the calcium indicator molecule is membrane permeable and capable of generating a detectable signal that is proportional to the level of calcium in the cytosol, with (ii) a calcium ionophore, under conditions and for a time sufficient to increase calcium levels within the cell; (b) detecting the signal generated by the calcium indicator molecule at a plurality of time points; (c) repeating steps (a) and (b) at least once; and (d) comparing (i) the signal generated by the calcium indicator molecule at one or more of the time points prior to and following at least one of the contacting steps in the absence of the candidate agent to (ii) the signal generated by the calcium indicator molecule at one or more of the time points prior to and following at least one of the contacting steps in the presence of the candidate agent, wherein an increased level of calcium in the cytosol at a time point prior to a contacting step in the presence of the agent, compared to the level of calcium in the cytosol prior to a contacting step in the absence of the agent, indicates an agent that uncouples oxidative phosphorylation from ATP production.
In another embodiment the invention provides a method of identifying an agent that is a respiratory inhibitor, comprising: (a) contacting (i) a biological sample comprising a cell containing cytosol, a mitochondrion and a calcium indicator molecule, under conditions that permit maintenance of mitochondrial membrane potential, and wherein the calcium indicator molecule is membrane permeable and capable of generating a detectable signal that is proportional to the level of calcium in the cytosol, with (ii) a calcium ionophore, under conditions and for a time sufficient to increase calcium levels within the cell; (b) detecting the signal generated by the calcium indicator molecule at a plurality of time points; (c) repeating steps (a) and (b) at least once; and (d) comparing (i) the signal generated by the calcium indicator molecule at one or more of the time points prior to and following at least one of the contacting steps in the absence of the candidate agent to (ii) the signal generated by the calcium indicator molecule at one or more of the time points prior to and following at least one of the contacting steps in the presence of the candidate agent, wherein an increased level of calcium in the cytosol at a time point prior to a contacting step in the presence of the agent, compared to the level of calcium in the cytosol prior to a contacting step in the absence of the agent, indicates an agent that is a respiratory inhibitor.
In another embodiment the invention provides a method of identifying an agent that alters a mitochondrial calcium uniporter, comprising (a) contacting (i) a biological sample comprising a cell containing cytosol, a mitochondrion and a calcium indicator molecule, under conditions that permit maintenance of mitochondrial membrane potential, and wherein the calcium indicator molecule is membrane permeable and capable of generating a detectable signal that is proportional to the level of calcium in the cytosol, with (ii) a calcium ionophore, under conditions and for a time sufficient to increase calcium levels within the cell; (b) detecting the signal generated by the calcium indicator molecule at a plurality of time points; (c) repeating steps (a) and (b) at least once; and (d) comparing (i) the signal generated by the calcium indicator molecule at one or more of the time points prior to and following at least one of the contacting steps in the absence of the candidate agent to (ii) the signal generated by the calcium indicator molecule at one or more of the time points prior to and following at east one of the contacting steps in the presence of the candidate agent, wherein an increased level of calcium in the cytosol at a time point following a contacting step in the presence of the agent, compared to the level of calcium in the cytosol following a contacting step in the absence of the agent, indicates that the agent alters a mitochondrial calcium uniporter.
It is another aspect of the invention to provide a method of identifying an agent that alters mitochondrial function, comprising (a) contacting (i) a biological sample comprising a permeabilized cell depleted of cytosol, a mitochondrion and a calcium indicator molecule, under conditions that permit maintenance of mitochondrial membrane potential, with (ii) a source of calcium cations, wherein the calcium indicator molecule is capable of generating a detectable signal that is proportional to the level of calcium in the cell; (b) detecting the signal generated by the calcium indicator molecule at a plurality of time points; and (c) comparing the signal generated by the calcium indicator molecule at one or more of the time points in the absence of a candidate agent, to the signal generated by the calcium indicator molecule at one or more of the time points in the presence of the candidate agent, and therefrom identifying an agent that alters mitochondrial function. In another related embodiment there is provided such a method in a high throughput screening format, wherein the step of contacting is performed in each of a plurality of reaction vessels in a high throughput screening array, and the step of detecting is performed in each reaction vessel.
According to certain further embodiments, the calcium indicator molecule is capable of generating a detectable signal that is proportional either to the level of calcium in the mitochondrion or to the level of calcium outside of the mitochondrion. In certain other further embodiments the step of contacting is repeated at least once. In certain other further embodiments the sample contains at least one compound that alters intracellular distribution of a calcium cation. In a still further embodiment the compound that alters intracellular calcium cation distribution is thapsigargin or Ru360. In another embodiment the sample contains at least one compound that uncouples oxidative phosphorylation from ATP production. In another embodiment the source of calcium cations is exogenous to the cell, and in another embodiment the sample contains at least one compound that uncouples oxidative phosphorylation from ATP production. In certain embodiments the cell comprises at least one polypeptide that is a Bcl-2 family member, and in certain other embodiments the cell expresses a gene encoding a polypeptide that regulates cytosolic calcium. In a further embodiment the gene encodes a mitochondrial calcium uniporter, and in a distinct further embodiment the gene is a transfected gene. In a still further embodiment the gene encodes a mitochondrial calcium uniporter. According to certain embodiments the cell adheres to a solid substrate, while in certain other embodiments the cell is a non-adherent cell.
In another embodiment the present invention provides a method of identifying an agent that alters mitochondrial function, comprising (a) contacting (i) a biological sample comprising one or more isolated mitochondria and a calcium indicator molecule in a medium, under conditions that permit maintenance of mitochondrial membrane potential, with (ii) a source of calcium cations, wherein the calcium indicator molecule is capable of generating a detectable signal that is proportional to the level of calcium in the biological sample; (b) detecting the signal generated by the calcium indicator molecule at a plurality of time points; and (c) comparing the signal generated by the calcium indicator molecule at one or more of the time points in the absence of a candidate agent, to the signal generated by the calcium indicator molecule at one or more of the time points in the presence of the candidate agent, and therefrom identifying an agent that alters mitochondrial function. In another related embodiment there is provided such a method in a high throughput screening format, wherein the step of contacting is performed in each of a plurality of reaction vessels in a high throughput screening array, and the step of detecting is performed in each reaction vessel.
In certain further embodiments the calcium indicator molecule is capable of generating a detectable signal that is proportional to the level of calcium in the mitochondrion, and in certain other further embodiments the calcium indicator molecule is capable of generating a detectable signal that is proportional to the level of calcium outside of the mitochondrion. In certain embodiments the step of contacting is repeated at least once. In another embodiment the sample contains at least one compound that alters distribution of a calcium cation in the sample, which in certain further embodiments is thapsigargin or Ru360. In another embodiment the sample contains at least one compound that uncouples oxidative phosphorylation from ATP production, and in another embodiment the isolated mitochondria are derived from a cell that comprises at least one polypeptide that is a Bcl-2 family member. In certain other embodiments the isolated mitochondria are derived from a cell that expresses a gene encoding a polypeptide that regulates cytosolic calcium, and in certain further embodiments the gene encodes a mitochondrial calcium uniporter. In certain other embodiments the gene is a transfected gene, and in certain further embodiments the gene encodes a mitochondrial calcium uniporter.
In certain further embodiments of invention methods described above, subsequent to the step of contacting the biological sample with the source of calcium cations and prior to the step of comparing signals, the biological sample is contacted (i) with at least one compound that uncouples oxidative phosphorylation from ATP production, and (ii) with at least one agent that alters mitochondrial function. In some embodiments the agent that alters mitochondrial function is cyclosporin A, and in certain other embodiments the agent is cyclosporin A, rotenone, oligomycin, succinate or Bcl-2. In certain embodiments the compound that uncouples oxidative phosphorylation from ATP production is FCCP or CCCP.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.